The S-acyl-2-mercaptobenzamide thioester (SAMT) inhibitors are low molecular weight compounds which target multiple steps in the HIV replication pathway, but primarily function to specifically inactivate cell-free HIV immediately upon exposure to the reactive compounds and to suppress the production of infectious HIV from virus-infected cells. These NCp7-targeted, virus inactivating compounds act by stripping coordinated zinc ions from the nucleocapsid (NC) protein in the infectious virion or maturing virus particle. In the process, the compounds irreversibly cross-link the nucleocapsid proteins rendering the virion noninfectious and defective. Thus, the NCp7 inhibitors interfere with two potential virus transmission mechanisms required for the infection of target cells in the vaginal environment. In the R21 phase of this proposal we propose to develop new microbicides composed of polymeric prodrugs for delivery of the SAMTs. This delivery mechanism limits the tissue absorption of the SAMT until it comes in contact with the viral inoculum in semen by attaching it to a high molecular weight biocompatible polymer. We will conjugate the SAMT inhibitors to the polymer carrier through enzyme-cleavable linkages that will release the active drug product in the presence of specific enzymes in semen. This delivery approach offers several advantages in the context of microbicide action since (1) the NCp7 inhibitors can inactivate cell-free and cell-associated virus in semen, we will target the virus before it can diffuse in an infectious form to or into tissue, (2) we will add moieties to the polymer backbone that will increase the stability of the SAMT inhibitors by decreasing the pH local to the conjugated drug by the Donnan effect, and (3) since microbicides will be used by women repeatedly over many years, a polymeric prodrug approach will allow precise control over the tissue concentrations and exposure to anti-HIV compounds, limiting the chance to develop viral resistance and limiting toxicity. Critical to the development of this prodrug approach, biological evaluations will be performed to confirm the efficacy of the SAMTs in the presence of seminal plasma and vaginal fluids. Additionally, the enzymatic activation of the compound from its prodrug form will be evaluated in specially designed in vitro assays to mimic the events which must occur in the vagina and to quantify the kinetics of drug activation and virus inactivation in the presence of semen and other appropriate biological matrices. Finally, the biological properties of both semen and vaginal fluids on the efficiency of transmission of HIV to target cells will be evaluated to define the potential synergies between the antiviral activity of constituents of semen and the biological activity of the thioester inhibitors.